Comparison of a monoclonal antibody-based capture/enrichment sandwich enzyme linked immunosorbent assay with immunomagnetic bead separation for the detection of attachment effacement Escherichia coli O26 strains from cattle faeces

Abstract

Aims: A monoclonal antibody (Mab 2F3)‐based sandwich enzyme linked immunosorbent assay (sELISA) format for the detection of Escherichia coli O26 that improves the sensitivity of the assay by combining enrichment with the capture stage has been developed. Culture of the enriched contents of wells before completion of the sELISA was compared with immunomagnetic bead separation (IMS) as a means of specific isolation of the target organism. Methods and Results: Bovine faecal samples, c. 10% in buffered peptone water (BPW), were pre‐enriched for 6 h before testing by capture/enrichment sELISA and by IMS. The sELISA consisted of a 1–2 h capture stage followed by addition of BPW to the wells and an overnight enrichment stage before completion of the assay. The capture/enrichment stage of the assay was repeated a second time on the enriched contents removed from the wells before completion of the first sELISA. From 204 cattle faeces samples, 30× O26 strains [20× attachment effacement Escherichia coli (AEEC) and 10× non‐AEEC] were isolated from the enriched wells of the sELISA, in comparison with 11 (9× AEEC and 2× non‐AEEC) that were isolated by IMS. Examination of the use of enterohaemolysin activity and rhamnose utilization on 1% rhamnose McConkey's (RMAC) agar with or without cefixime and potassium tellurite demonstrated that the selection based on enterohaemolysin production and growth on RMAC with cefixime and potassium tellurite would largely differentiate the AEEC strains from the non‐AEEC strains. Conclusions: The capture/enrichment sELISA protocol used compared favourably with the IMS for the isolation of E. coli O26 from faeces samples. The ELISA optical density readings obtained in the procedure were used as a screening indicator for selection of samples for further culture examination, and the selective culture methods examined to assist strain isolation did have potential. Significance and Impact of the Study: The capture/enrichment format of an Mab‐based sELISA protocol has the potential to provide a suitable screening assay for the specific detection of pathogenic strains from mixed culture samples like faeces.

Escherichia coli serogroup O26 ? a new look at an old adversary

“…ELISA detecting the presence of vtx 1 and vtx 2 have been used to detect VTEC (Paton and Paton 2003), and more recently, monoclonal antibodies against O26 surface antigens have been used in an ELISA format for the specific detection of E. coli O26 (Neely et al. 2004) and compared favourably with IMS (Finlay et al. 2006).…”